In mammals, glucuronidation is a principle means of detoxifying or inactivating compounds which utilizes the UDP glucuronyl transferase system. In humans, a number of hormones, including cortisol and aldosterone testosterone and androsteindione, certain antibiotics such as chloramphenicol, toxins such as dinitrophenol, and bilirubin are among the compounds which are conjugated to form glucuronides by the glucuronyl transferase system and then excreted in urine or into the lower intestine in bile. The bacterium Escherichia coli has evolved to survive in the mammalian intestine, and can utilize the excreted .beta.-glucuronides as its sole carbon source. To do so, E. coli has evolved mechanisms for the uptake and degradation of a wide variety of glucuronides, processes which are tightly linked genetically.
2.1. Beta-Glucoronides
Most aromatic and aliphatic glucuronides are remarkably stable relative to other types of glycoside conjugates. It is speculated that this is due to the inductive effect of the carbonyl group at C-6 on the hemiacetal linkage at C-1. Many .beta.-glucuronides can be prepared free of other contaminating glycosides by vigorous acid hydrolysis, which cleaves glucosides, galactosides and other glycosides, but leaves most glucuronides intact. For example, complex carbohydrate polymers such as gum arabic can be reduced to a collection of monosaccharide components, and the single .beta.-glucuronyl disaccharide aldobiuronic acid, simply by boiling gum arabic in sulfuric acid overnight. Colorigenic and fluorogenic glucorogenic substrates such as p-nitrophenyl .beta.-D-glucuronide and 4-methylumbelliferyl .beta.-D-glucuronide are much more stable in aqueous solution than the corresponding .beta.-D-galactosides or .beta.-D-glucosides.
.beta.-Glucuronides in polysaccharide form are common in nature, most abundantly in vertebrates, where in polymeric form with other sugars such as N-acetylglucosamine they are major constituents of connective and lubricative tissues (e.g. chondroitin sulfate of cartilage, and hyaluronic acid, the principle constituent of synovial fluid and mucus). .beta.-glucuronides are relatively uncommon in plants. However, some plant gums and mucilages produced by wounded trees, notably gum arabic from Acacia senegal, do contain significant fractions of .beta.-glucuronides in polymeric form, although rarely if ever as terminal residues. Glucuronides and galacturonides found in plant cell wall components (such as pectin) are generally in the alpha configuration, and are frequently substituted as the 4-O-methyl ether; hence these are not substrates for .beta.-glucuronidase.
As simple glycosides, .beta.-glucuronides are extremely important as the principle form in which xenobiotics and endogenous phenols and aliphatic alcohols are excreted in the urine and bile of vertebrates (reviewed by Dutton, G. J., 1966, ed., Glucuronic Acid, Free and Combined, NY; Dutton, G. J., 1980, Glucuronidation of Drugs and Other Compounds, Florida). Detoxification of xenobiotics by glucuronidation is the most important mechanism for elimination of inappropriate compounds from the metabolism of vertebrates. Glucuronidation occurs in many tissues in vertebrates, most notably in the liver. The reaction is carried out by a set of membrane-bound enzymes catalyzing the transfer of a glucuronate residue from uridine diphosphate 1-alpha-D-glucuronate to the aglycone (xenobiotic). There are several isozymes of UDP-glucuronyl transferase that have been characterized (for a thorough review, see Dutton, 1980, supra). These enzymes are frequently part of a suite of detoxifying enzymes, including hydroxylases and mixed-function oxidases that work in concert to metabolize lipophilic, relatively insoluble compounds into the highly water-soluble glucuronide conjugates (as well as sulfates and other derivatives). These conjugates are then excreted into the bile (for the larger glucuronide conjugates) or the urine. Several thousand .beta.-glucuronides have been characterized in urine and bile as detoxification products, many following administration of the free aglycone or a related compound (a compendium of many glucuronides can be found in Dutton, 1966, supra). In addition, many endogenous steroid hormones and bioactive substances, or bio-degradation products such as bilirubin, are conjugated and excreted as .beta.-glucuronide conjugates. This extremely important and voluminous pathway and the fact that, among enteric bacteria, the .beta.-glucuronidase gene appears almost exclusvely limited to E. coli may account in part for the success of E. coli as a principle and ubiquitous colonizer of the vertebrate intestine and urinary tract.
Interestingly, .beta.-glucuronidase activity is reliably reported almost exclusively from those organisms that have, or are associated with organisms that have glucuoridation as a detoxification pathway. Thus vertebrates, which all use glucuronidation as the principle conjugation mechanism, together with some of their endogenous microbe populations (usually E. coli) have GUS activity. By contrast, insects and plants conjugate xenobiotics with glucose, rather than glucuronic acid, as their detoxification and derivatization mechamism, and .beta.-glucuronidase is rarely if ever reported in these organisms or their attendant microbial populations.
2.2. Beta-Glucuronidase
Beta (.beta.)-glucuronidase (GUS) catalyzes the hydrolysis of a very wide variety of .beta.-glucuronides, and, with much lower efficiency, hydrolyzes some .beta.-galacturonides; the reaction and a small selection of the available substrates for routine assay of the enzyme are diagrammed in FIG. 13. Almost any aglycone conjugated in a hemiacetal linkage to the C1 hydroxyl of a free D-glucuronic acid in the B configuration serves as a GUS substrate. Glucuronides are generally very water soluble, due to the ionizable carboxylic acid group at the 6-carbon position in the glycone.
E. coli .beta.-glucuronidase (GUS) has a monomer molecular weight of about 68,200 daltons, although under certain conditions of SDS-polyacrylamide gel electrophoresis it migrates a bit slower than would be predicted (around 72-74 kDa). The behaviour of the native enzyme on gel filtration columns indicates that the active form is probably a tetramer. It is not processed at the amino terminus in E. coli, and is found exclusively in the cytoplasm. GUS is an exo-hydrolase; it will not cleave glucuronides in internal positions within polymers. GUS is specific for .beta.-D-glucuronides, with some tolerance for .beta.-galacturonides. It is inactive against .beta.-glucosides, .beta.-galactosides, .beta.-mannosides, or glycosides in the alpha configuration.
.beta.-Glucuronidase is very stable, and will tolerate many detergents and widely varying ionic conditions. It is most active in the presence of thiol reducing agents such as .beta.-mercaptoethanol or dithiothreitol (DTT). GUS has no cofactors, nor any ionic requirements. GUS is inhibited by some divalent metal ions: 70% inhibition by Mn.sup.2+ and Ca.sup.2+ at 10 mM, and completely by Cu.sup.2+ and Zn.sup.2+ at comparable concentrations (Stoeber, 1961, Etudes des propietes et de la biosynthesase de la glucuronidase et de la glucuronide-permease chez E. coli. These de Docteur es Sciences, Paris). .beta.-Glucuronidase can be assayed at any physiological pH, with an optimum between 5.0 and 7.8. The enzyme is about 50% as active at pH 4.3 and at pH 8.4. GUS from E. coli K12 is reasonably resistant to thermal inactivation with a half-life at 55.degree. C. of about two hours and at 60.degree. C., about 15 minutes. The specific inhibitor, glucaric acid 1,4 lactone (saccharic acid lactone, saccharolactone) is a very useful reversible competitor inhibitor of GUS.
.beta.-Glucuronidase activity is extremely common in almost all tissues of all vertebrates and many molluscs (Levvy, G. A. and Conchie, J., 1966, .beta.-Glucuronidase and the hydrolysis of glucuronides, in Glucuronic Acid, Free and Combined, NY. p. 301). The enzyme has been purified from many mammalian sources (e.g. Tomino et al., 1975, J. Biol. Chem. 250:8503) and shows a homotetrameric structure, with a subunit molecular weight of approximately 70 kDa. The enzyme from these sources is synthesized with a signal sequence at the amino terminus, and is then transported to and glycosylated within the endoplasmic reticulum and ultimately localized within vacuoles intracellularly. Unlike the bacterial enzyme, mammalian and molluscan GUS can cleave thioglucuronides. In general, however, the E. coli GUS is much more active than the mammalian enzyme against most biosynthetically derived .beta.-glucuronides (Tomasic, J. and Keglevic, D., 1973, Biochem. J. 133:789-795; and Levvy and Conchie, supra). The genetics of GUS in mammals have been extensively characterized (reviewed in Paigen, K., 1979, Ann. Rev. Genet. 13:417-466).
GUS activity is largely if not completely absent from higher plants (Jefferson et al., 1987, EMBO J. 6:3901-3907) mosses, algae and ferns. There are a few reports of endogenous activity in plants but they rarely include quantitative tests with more than one substrate, to ensure that the activity is a true .beta.-glucuronidase, not an activity specific for the aglycone of the test substrate (e.g., Schultz, M. and Weissenbock, G., 1987, Phytochemistry 26:933-938), nor do they often make use of specific inhibitors of GUS such as saccharolactone (see below). Such reports should also be interpreted cautiously because only rarely do plants exist without numerous exo- and endophytic organisms, many not yet classified, which could be contributing GUS activity. Specific glucuronidase that recognize endogenous substrates such as glycyrrhizin conjugates have been described, but are not capable of cleaving GUS assay substrates.
The free-living soil nematode, Caenorhabditis elegans, has an endogenous .beta.-glucuronidase activity which occurs at low levels in the intestine of the worm. Enzyme activities in the other nematodes have apparently not been investigated.
Very few insects have been investigated for intrinsic GUS activity. Studies on Drosophila melanogaster embryos, pupae and larvae showed no detectable activity under conditions that gave very high levels of .beta.-galactosidase (Jefferson, 1985, (published 1986) DNA Transformation of Caenorhabditis elegans: Development and Application of a New Gene Fusion System. PhD. Dissertation, University of Colorado, Boulder). Extracts from white flies and black flies from glasshouse populations also revealed very little if any GUS activity. Locust crop fluid liquor is a source of GUS but it is not clear whether this is an intrinsic activity, or due to microorganisms in the crop fluid.
GUS activity has not yet been found in any fungi, including Saccharomyces, (Jefferson, 1985, DNA Transformation of Caenorhabditis elegans: Development and Application of a New Gene Fusion System. PhD. Dissertation, University of Colorado, Boulder; and Schmitz et al., 1989, Gene, in press) Schizosaccharomyces, Aspergillus, Neurospora, Cladosporium, Leptosphaeria and other Ascomycetes such as barley powdery mildew or Oomycetes such as Bremia lactuca. There is also no detectable activity in the slime mould, Dictyostelium discoidium (Datta et al., 1986, Molec. Cell. Biol. 6:811-820; and Jefferson, 1985, DNA Transformation of Caenorhabditis elegans: Development and Application of a New Gene Fusion System. PhD. Dissertation, University of Colorado, Boulder).
GUS is not present in most bacterial genera examined, including Bacillus, Klebsiella, Proteus, Erwinia, Rhizobium, Bradyrhizobium, Agrobacterium, Pseudomonas, Xanthomonas, Anabaena and Actinomycetes although it must be remembered that induction of genes for GUS is required before activity can be found even in definitively GUS.sup.+ bacteria. The intestinal commensal Enterobacteriaceal species E. coli is one of the only species of bacteria that has been found reliably to have a .beta.-glucuronidase activity; in fact, the presence of .beta.-glucuronidase is a widely accepted diagnostic test for E. coli in natural populations of bacteria isolated from sources such as urine, feces, contaminated water or food (e.g. Godsey, et al., 1981, J. Clin. Microbiol. 13:483-490; Feng, P. C. S. and Hartman, P. A., 1982, Appl. Environ. Microbiol. 43:1320-1329; Trepeta, R. W. and Edberg, S. C., 1984, J. Clin. Microbiol. 19:172-174; and Moberg, L. F., 1985, Appl. Environ. Microbiol. 50:1383-1387). The GUS activity of E. coli populations in the intestine plays a very significant role in the physiology of most vertebrates, being partially or wholly responsible for enterohepatic recirculation of conjugated drugs, hormones and xenobiotics. Recent work indicates that there is at least one other genus of bacterium (Alcaligenes sp.) found in soil and water samples, and in urine and feces, that appears to produce GUS upon induction. Regions of the E. coli chromosome, containing portions of the GUS operon have been subcloned into E. coli plasmids (Blanco et al., 1982, J. Bacteriol. 149:587-594); however, prior to the present invention, the gene encoding GUS had not been isolated and characterized, nor had the coding system been expressed in a heterologous system.
In addition to .beta.-glucuronidase enzyme, the GUS operon of E. coli also encodes glucuronide permease, first described biochemically by F. Stoeber (1961, These de Docteur des. Sciences, Paris). Glucuronide permease provides a mechanism for transport of .beta.-glucuronidase through the cell membrane, and permits the entrance of a surprisingly wide variety of substrates, ranging from simple aliphatic compounds to large, complex heterocyclic conjugates (FIG. 13), into the cytoplasm. .beta.-galactoside permease, in contrast, will only admit very simple molecules but not compounds of any appreciable complexity (e.g. complex phenolic compounds or heterocyclic compounds such as x-gal.
The combination of GUS and glucuronide permease enables E. coli to utilize a vast repertoire of glucuronides as sources of energy. Numerous compounds (including hormones, cholesterol, and antibiotics) are conjugated to glucuronic acid in the human liver, and thereby nourish the bacteria that constitute the intestinal flora. In turn, by metabolizing these compounds, E. coli significantly impacts on the bioavailability of a multitude of biologically relevant molecules.
2.3. The Utility of Gene Fusion Systems
"Reporter" genes are used in molecular biology as indicators of gene activity. A reporter gene will typically encode ah enzyme activity that is lacking in the host cell or organism which is to be transformed. This allows the measurement or detection of the enzyme activity which may be used as an indicator or "reporter" of the presence of expression of the newly introduced gene.
A reporter gene may be put under the influence of a "controller" sequence, such as a promoter element. Successful expression of reporter gene product serves as an indicator of controller element activity. Additionally, a reporter gene may be used as a DNA transformation marker. Cells may be transformed with DNA comprising a gene of interest which encodes a product that is difficult or impossible to detect as well as DNA comprising the reporter gene under the control of a suitable transcriptional promoter; expression of reporter gene activity in a cell is suggestive of successful transformation with the gene of interest, the presence of which may be corroborated by standard molecular techniques. Measurement of reporter enzyme activity is frequently used to infer characteristics of the transcription of a gene encoding the reporter enzyme. These inferences depend on several assumptions that should be examined, and, when possible, controlled experimentally. Firstly, we should be aware that the ultimate regulated level of a gene product is determined by a large number of factors, only one of which is the initiation of transcription. While transcription does appear to be the principle site of regulation of gene action, there are numerous other components in the regulatory pathway that must be considered, and in some situations they will prove to be more important than transcriptional control. For instance, it is clear that DNA modifications such as methylation, chromatin configuration and possibly three dimensional structure and location of the gene can influence its expression. It is also obvious that control of precursor RNA processing and transport--including the correct excision of introns, polyadenylation of the transcript, extranuclear transport to a site of translation, and degradation of the mRNA or its precursors can have profound effects on the eventual levels of a protein product. The frequency of translational initiation, the rate of extension, the processing, modification and/or targeting or the primary protein product as well as its degradation and turnover will also inevitably affect final product levels.
The use of precise gene fusions can simplify analysis of this complex process. For example, it is possible to delineate the contribution of transcriptional control of gene expression by eliminating all the specific signals for post-transcriptional controls and replacing them with sequences from a readily assayed responder gene. Further careful gene fusion constructions can then be performed to assay the effects of inclusion of additional controller sequences, for instance the "untranslated leader sequences" or the sequences surrounding the site of translational initiation. Gene fusions need not be confined to promoter analysis, since factors affecting mRNA processing and stability, (such as polyadenylation signals or introns), or translational efficiency (such as the context of the initiator codon or mRNA secondary structure), will inevitably affect reporter enzyme levels. With the appropriate controls, many of these regulatory steps can also be analyzed with gene fusion technology. It is important to be aware of the potential contributions of these "downstream" points in the regulatory pathway of gene expression so they can be considered in the design and interpretation of gene fusion experiments.
In addition, many genes in plants and other higher organisms exist in multi-gene families whose products are very similar but can be regulated differentially during development; in fact many times members of multi-gene families are apparently inactive. By using gene fusions to individual members of such families and introducing these fusions into the genome, one can study the expression of individual genes separate and distinct from the background of the other members of the gene family.
Analysis of mutationally altered genes in plants accessible to transformation techniques is greatly facilitated by the use of sensitive and versatile reporter enzymes. Many of the regulatory parameters responsible for spatial and temporal restriction of gene activity require specialized analytic methods. Moreover, the logistics of analyzing gene function in large numbers of transgenic plants can be overwhelming, unless routine, high resolution techniques are available. Although many of the plant genes that have been characterized to date produce abundant products that are measurable by existing means, many more will certainly be described whose products are of moderate or low abundance; these will doubtlessly prove important and interesting to study, requiring increasingly sensitive methods. By using a reporter gene that encodes an enzyme activity not found in the organism being studied, the sensitivity with which chimeric gene activity can be measured is limited only by the properties of the reporter enzyme and the quality of the available assays for the enzyme.
2.3.1. A Review of Existing Gene Fusion Systems
An ideal gene fusion system should provide a reporter enzyme that is stable, tolerates amino terminal fusions, has numerous, simple and versatile assays, and that has no intrinsic background activity in the organism being studied. Furthermore, the enzyme should not interfere with normal physiological functioning of the organism, nor affect the biochemistry adversely. The assays should be sensitive enough to measure gene expression of moderate to low abundance in single cells, and should allow spatial discrimination of enzyme activity within the complex cellular patterns of tissues and organs. In addition, the assays should be quantitative, inexpensive and uncomplicated. The enzyme should be active under widely varying conditions of pH, ionic environment and temperature, and should be tolerant of general laboratory manipulations. There should also be the possibility of using the system as a true responder, providing both reporting and effecting functions, thereby allowing genetic selections to be applied. There should be methods available to use the system in live organisms quantitatively. Progress in agricultural molecular biology, and especially the use of gene fusions in transgenic plants, fungi and bacteria of agricultural and industrial importance will be greatly enhanced by the availability of suitable responder genes encoding enzymes with this set of properties.
At least seven reporter genes have been used in studies of gene expression in higher plants. These include the E. coli .beta.-galactosidase (lacZ, LAC), chloramphenicol acetyl transferase (CAT), neomycin phosphotransferase (APH3'II, NPTII), nopaline synthetase (NOS), octopine synthase (OCS), firefly luciferase (luc) and bacterial luciferase (luxA and luxB). Each of these systems has properties that make them less than optimal for gene fusion analysis.
The lacZ gene from E. coli is part of an operon of three genes--the lac operon--and encodes a stable .beta.-galactosidase with a wide substrate specificity. This gene was the first used in gene fusion experiments in the construction of the trp-lac fusion in the E. coli chromosome, well before the advent of recombinant DNA manipulations in vitro (Beckwith et al., 1967, Transposition of the lac region of E. coli, Cold Spring Harbor Symp. Quant. Biol. 31:393.; Miller et al., 1970, J. Bacteriol. 104:1273) and has been very widely used in studies in E. coli and other bacteria, and somewhat in fungi and animals. LAC fusions have been a very powerful tool due to the detailed genetic, biochemical and molecular understanding of the operon and its encoded proteins and to the availability of selective substrates such as lactose as well as substrates for spectrophotometric, fluorometric and histochemical assays. In addition, the wide availability of E. coli strains deficient in the components of the lac operon has enhanced its implementation in E. coli.
In spite of the remarkable success of lacZ fusions in the development of E. coli molecular genetics, .beta.-galactosidase fusions (Helmer et al., 1984, Bio/technology 2:520-527) are difficult if not impossible to use effectively in plants because of very high endogenous .beta.-galactosidase activity. .beta.-Galactosidases are present in virtually all plants, and in most, if not all tissues. They are also present in many bacteria and fungi of agricultural and biotechnological importance. Additionally, intrinsic .beta.-galactoside compounds exist in all these organisms that could be degraded by the introduction of a .beta.-galactosidase activity, hence altering the physiology of the organism. It is possible in at least one plant system to selectively reduce or eliminate endogenous .beta.-galactosidase background activity under conditions for histochemical analysis leaving some bacterial .beta.-galactosidase activity. However, this treatment is not general, must be calibrated for each plant system, offers no quantitative methods, and hence is unlikely to open significant new prospects.
The Agrobacterium tumefaciens Ti-plasmid-encoded genes nopaline synthase (Depicker et al., 1983, J. Molec. Applied Genetics 1:561-575; Bevan et al., 1983a, Nature 304:184-187) and octopine synthase (De Greve et al., 1982, J. Mol. Applied Genetics, 1:499-513) have been used as reporter genes in the past because the opines produced by these enzymes are not normally found in plant cells. Moreover the genes were readily available, and are routinely transferred to plants upon Agrobacterium infection. However, these reporter genes are no longer widely used because the assays are cumbersome, of limited specificity, difficult to quantitate (Otten et al., 1978, Biochem. Biophys. Res. Commun. 527:497-500) and give no spatial information. In addition, octopine synthase cannot tolerate amino-terminal fusions (Jones et al., 1985, EMBO J. 4:2411-2418).
Until recently, the two most widely used reporter genes have been the bacterial genes chloramphenicol acetyl transferase (CAT) and neomycin phophotransferase (NPTII) which encode enzymes with specificities not normally found in plant tissues (Hererra-Estrella et al., 1983a, Nature 303:209-213; Fraley et al., 1983, J. Histochem, Cytochem. 13:441-447).
CAT catalyzes the transfer of an acetate group from acetyl-coenzyme A to one or both of the free hydroxyl groups of the antibiotic chloramphenicol, thus rendering it pharmacologically inactive. The gene and its encoded enzyme have been well characterized and the enzyme is quite stable. The most common assays involve incubation of an extract with limiting concentrations of radioactively labelled chloramphenicol and excess acetyl CoA, followed by organic extraction of the reaction products, which are more hydrophobic than the substrate, separation of the products on thin layer chromatograms and resolution of the incorporated radioactivity by autoradiography. Quantitation of the radioactivity is then done by excision of the spots from the TLC and liquid scintillation counting. This is a relatively expensive and cumbersome assay (Gorman et al., 1982, Mol. Cell. Biol. 2:1044). Alternative assays have been developed using radiolabelled acetyl CoA that avoid thin layer chromatography (Tomizawa, 1985, Cell 40:527-535; and Sleigh, 1986, Anal. Biochem, 156:251-256) but these are also expensive and prone to difficulties in extrapolating from quantitation of incorporated acetate to enzyme concentrations. Recent developments using HPLC, or fluorescently labeled chloramphenicol have streamlined this process somewhat.
NPTII, also called APH 3'II, catalyses the transfer of the terminal phosphate group from ATP to the antibiotic neomycin and its analogs, including geneticin (G418), and kanamycin. Chromatographic or electrophoretic assays have been developed to detect the activity by monitoring .sup.32 P incorporation into the antibiotic after incubating extracts with terminally .sup.32 P labelled ATP and substrate (e.g., Reiss et al., 1984, Gene 30:217-223). NPTII can tolerate amino-terminal fusions and remain enzymatically active, making it useful for studying transport into organelles in plants.
Both CAT and NPTII are relatively difficult, tedious and expensive to assay and suffer from variable endogenous activities in plant and animal cells (generally caused by enzymes with broader substrate specificity), which limits their sensitivity. Competing reactions catalyzed by endogenous esterases, phosphatases, transferases and other enzymes also make quantitation of CAT or NPTII by enzyme kinetics very difficult, and quantitation without enzyme kinetics to be very suspect. In addition, there are no reasonable methods for identifying cell or tissue localization with these enzymes. Because NPT II is a very versatile antibiotic selection marker (an effector) for transformation of plants, it will probably continue to be used in this capacity for some time. However, its use as a reporter gene may be ephemeral.
Attention has recently focussed on methods for light production in genetically engineered organisms using either of two luciferase genes. The firefly luciferase gene has been used as a marker in transgenic plants (Ow et al., 1986, Science 234:856-859), but the enzyme is labile, and is difficult and expensive to assay with accuracy (for a good review of the difficulties see DeLuca, M. and McElroy, W. D., 1978, Methods in Enzymology 57:3-15), the reaction is complex and there may be little potential for routine, affordable and meaningful histochemical analysis or fusion genetics. The genes luxA and luxB from Vibrio harveyi have also been used in several studies to monitor gene action in transgenic plants (Koncz et al., 1987, Proc. Natl. Acad. Sci., USA 84:131-135; and Langridge et al., 1989, Proc. Natl. Acad. Sci. USA 86:3219-323). However, detection of light production in situ by the expression of gene fusions of luxA and luxB, or a fused luxAB requires rather sophisticated single-photon capture and imaging systems may not unlikely to be affordable in the foreseeable future to most laboratories. In addition, there are currently no available methods to alter the spectral output to overcome absorption by the sample.
Although there is the assertion, frequently made, that the firefly and bacterial luciferases offer the possibility of true in vivo analysis, these claims should be examined carefully. First both systems require the uptake of exogenous substrates into living cells--in the case of the firefly luciferase, a complex charged molecule--in the case of the bacterial lux gene products, a relatively simple volatile aldehyde. To produce light, however, these compounds must then interact with the enzyme (modified and targeted to glyoxysomes in the case of the firefly enzyme, a heterodimer of two gene products in the case of the bacterial luciferase) and with cofactors and other substrates: for firefly luciferase Mg.sup.2+, O.sub.2, ATP; for bacterial lux FMNH. The production of light will therefore depend on high concentrations of not only the exogenously applied substrate (a difficult task to attain or measure reliably and reproducibly in all cell types) but also non-limiting levels of the other co-factors and substrates. In fact, the assay for firefly luciferase is so sensitive to ATP concentrations that it is widely used to measure ATP levels in cells and extracts. While this is an interesting and important use of luciferase, it has only confounding effects on the use of luciferase as a gene fusion marker. Because of the complexity and expense of these systems and the increasingly apparent need to quantitate gene action in situ, it is unlikely that the light production methods will receive wide acceptance in the near future. It must always be recalled that the ultimate aim of quantitation, as regards gene fusion experiments, is not to quantitate photons or dye deposition but rather gene activity.